Maleylated-BSA suppresses IFN-gamma-mediated Ia expression in murine peritoneal macrophages

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.     

Regulation of I-A expression by murine peritoneal macrophages: differences linked to the Bcg gene

We previously reported that Mycobacterium bovis (strain BCG) induces continuous I-A expression when injected into BCG-resistant strains of mice. We have extended this observation by showing that Corynebacterium parvum also induces continuous I-A expression by macrophages from BCG-resistant but not BCG-susceptible mice. We have linked continuous expression to BCG resistance by using C.D2Ityr mice, which are congenic with BCG-susceptible BALB/c mice except for genes on a portion of chromosome 1, which contains the gene(s) for BCG resistance. Macrophages from C.D2Ityr mice continuously expressed I-A, whereas macrophages from BALB/c mice transiently expressed I-A. Continuous expression by macrophages from both Bcgr and Bcgs mice could be induced in vitro with rIFN-gamma. However, the continuous expression of I-A by macrophages from Bcgs mice required the continued presence of IFN-gamma, whereas that by Bcgr macrophages did not. The continuous expression of I-A by macrophages from Bcgs mice was also inhibited by hydrocortisone, cyclohexamide, tunicamycin, and monensin, whereas I-A expression by Bcgr macrophages was not affected. The continuous expression of I-A by macrophages from Bcgr mice did not require its continued synthesis. The significance of these findings to the induction of immunity and to antimicrobial resistance are discussed.     

Persistent expression of Ia-antigen on a subpopulation of murine resident peritoneal macrophages

The stability of Ia-antigen expression on murine resident peritoneal macrophages was assessed during the course of in vitro culture. Contrary to published findings with radioimmunoassays and immunofluorescence assays, the cultured cells bore Ia-antigen, as shown by their rosetting with sheep erythrocytes coupled with anti-Ia.2 monoclonal antibody. In support of this finding, cultured cells presented the copolymer of glutamine, alanine, and tyrosine (GAT) to GAT-primed T lymphocytes in an Ia-dependent manner. Thus, functional Ia antigen is present on cultured macrophages. Disappearance of the antigen after fixation of macrophages with either glutaraldehyde or paraformaldehyde, a routine procedure in the radioimmunoassays and immunofluorescence assays, explains its presumed absence on cultured cells.     

Effects of endotoxic shock in several functions of murine peritoneal macrophages

Gram negative sepsis and septic shock continue to be a major medical problem, with a complex physiopathology and it is associated with high mortality. Although secretion of cytokines such as tumor necrosis factor-alpha by macrophages is the principal host mediator of septic shock, other characteristic functions of macrophages implicated in their phagocytic capacity have not been studied in the process of endotoxic shock. In the present study we have used an intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg) in order to obtain an endotoxic shock model in adult female BALB/c mice. Peritoneal cell suspensions were obtained at several times (2, 4, 12 or 24 h) after injection and the following functions were studied on the peritoneal macrophages: adherence to substrate, mobility (spontaneous and directed or chemotaxis), ingestion of particles and superoxide anion production. The results showed a stimulation of adherence, ingestion and superoxide production as well as a decrease of chemotaxis in the animals injected with LPS. These effects changed with time after LPS injection. Thus, the increase of adherence and the decrease of mobility were higher during the first hours, whereas the increase in ingestion and superoxide production turned larger with time.     

IL-4 and IFN-gamma up-regulate substance P receptor expression in murine peritoneal macrophages

While the ability of macrophages to express authentic substance P receptors (i.e., NK-1 receptors) has been inferred from radioreceptor binding assays and functional assays and, most recently, by identification of NK-1 receptor mRNA expression, we know little about NK-1 expression at the protein level or what host factors might up-regulate expression of this receptor. In the present study we demonstrate that the cytokines IL-4 and IFN-gamma can increase the expression of NK-1 receptors on murine peritoneal macrophages. Specifically, we show that IL-4 and IFN-gamma can elicit increases in the level of mRNA encoding the NK-1 receptor by up to 12- and 13-fold, respectively. Furthermore, these cytokines can significantly increase the expression of the NK-1 receptor protein as measured by Western blot and FACS analysis using specific Abs developed in our laboratory. In addition, we have demonstrated the ability of both IL-4 and IFN-gamma to enhance the ability of macrophages to bind substance P as measured by radiolabeled binding assay. The observation that the level of expression of this receptor protein can be enhanced by cytokines that promote either cell-mediated (Th1) or humoral (Th2) immune responses supports the idea that this receptor can be induced during either type of immune response. As such, these results may point to a more ubiquitous role for substance P in the generation of optimal immune responses than previously appreciated.     

Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus

Protein expression changes induced in thioglycolate‐elicited peritoneal murine macrophages (MΦ) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control MΦ and MΦ incubated 2 h with live or heat‐inactivated GBS were separated by 2‐DE. Proteins whose expression was significantly different in infected MΦ, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in MΦ by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS‐infected MΦ. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose‐cycle enzymes being down‐regulated in MΦ infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.     

Effects of sexual steroid hormones on the functionality of murine peritoneal macrophages.

In the present work, we studied the possible effect of steroid hormones, estradiol, progesterone, and 5 alpha-dihydrotestosterone, on different phenotypic and functional characteristics of peritoneal adherent mononuclear cells. We used female and male mice of Balb/c strain, normal, gonadectomized, and gonadectomized with hormonal replacement. We found that gonadectomy in both sexes produced a significant decrease in the functionality of membrane receptors for the complement and in phagocytic activity of Candida albicans-anti-C albicans system. In addition, the percentages of cells that reduced nitroblue tetrazolium were diminished in castrated animals. Ovariectomized females injected with estradiol presented normal levels of phagocytic and metabolic capacities, but the expression of membrane receptors for complement remained decreased. In contrast, progesterone treatment of ovariectomized animals had the opposite effect. Simultaneous treatment with estradiol plus progesterone gave results similar to those observed with estradiol only. Dihydrotestosterone per se did not affect any of the parameters measured in the conditions used here. These results suggest that female steroids affect macrophage functionality, probably by regulating surface receptors that are involved in phagocytic activity.     

Amphotericin B inhibits the serum-induced expression of tissue transglutaminase in murine peritoneal macrophages

Culture of mouse resident peritoneal macrophages (PM) in serum-containing medium causes a rapid and marked induction of the enzyme tissue transglutaminase (tissue TGase). Coculture of PM with amphotericin B (AmpB) inhibited the serum-induced expression and accumulation of tissue TGase. The AmpB-mediated inhibition of tissue TGase was specific and was due to inhibition of enzyme synthesis. The serum-dependent induction of tissue TGase was inhibited in a dose-dependent fashion, and a complete inhibition was observed at 1.5 microgram/ml dose of AmpB. The inhibition was reversible; however, the time of recovery depended on the dose and time of exposure of the cells to AmpB. The present studies suggest that AmpB-mediated inhibition of tissue TGase is due to inhibition of the uptake of serum retinoids by PM.     

Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages

The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappaB (NF-κB) signaling pathway of murine peritoneal macrophages. Since glutamine is essential for the normal functioning of macrophages, it was hypothesized that in vitro glutamine supplementation would increase NF-κB activation. Peritoneal macrophages were pretreated with glutamine (0, 0.6, 2 and 10 mM) before incubation with lipopolysaccharide (LPS), and the effects of glutamine on the production of tumor necrosis factor-alpha and on the expression and activity of proteins involved in the NF-κB signaling pathway were studied by an enzyme linked immuno-sorbent assay, Western blotting, and an electrophoretic mobility shift assay. Glutamine treatment (2 and 10 mM) increased the activation of NF-κB in LPS-stimulated peritoneal macrophages (P < 0.05). In non-stimulated cells, glutamine treatment (2 and 10 mM) significantly reduced IκB-α protein expression (P < 0.05). Glutamine modulates NF-κB signaling pathway by reducing the level of IκB-α, leading to an increase in NF-κB within the nucleus in peritoneal macrophages.     

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.     

Effects of cationic vectors complexed with plasmid DNA on the phagosome-lysosome fusion in murine peritoneal macrophages and J744 macrophages

Polyethylenimine (PEI) and cationic polypeptides complexed with plasmid DNA are the most efficient nonviral vectors for gene therapy. It is believed that endocytosis is the major pathway for cell entering by PEI/DNA or cationic peptides/DNA complexes. Effects of plasmid DNA complexed with PEI, poly-L-lysine (PLL), poly-D-lysine (PDL) and polyarginine (PA) on the phagosome-lysosome fusion (P-LF) were studied in murine peritoneal macrophages and J774 macrophages. Cationic polypeptide PLL can be hydrolysed by cellular peptidases, but its stereoisomer, PDL, cannot be split by these enzymes. PEI, PDL, and PA have been shown to inhibit P-LF. PLL showed a low effect on the P-LF. On the basis of these studies, we assume that lysosomotropic agents able to change functions of lysosomes in the cell may affect transfection efficiency and thus be used for gene therapy.     

Fc-receptor mediated protein phosphorylation in murine peritoneal macrophages

The effect of Fc receptor engagement on protein phosphorylation in murine peritoneal macrophages has been investigated. Treatment of macrophage cultures with insoluble immune complexes resulted in enhanced phosphorylation of six proteins at 73, 66, 53, 37, 31 and 25 kD. Comparison of the protein phosphorylation patterns induced by immune complexes with those induced by agents which mimic the actions of well known intracellular second messengers (i.e., A23187, dibutyryl cAMP, or phorbol myristate acetate) revealed substantial similarity between Fc receptor induced events and those induced in response to phorbol diesters. There were, however, two phosphorylated proteins which were only seen following stimulation with immune complexes. Thus, more than one kind of protein kinase activity appears to be involved in Fc receptor mediated stimulation of macrophage function.